Exploration of Resveratrol as a Potent Modulator of α-Synuclein Fibril Formation

Abstract

The molecular determinants of amyloid protein misfolding and aggregation are key for the development of therapeutic interventions in neurodegenerative disease. Although small synthetic molecules, bifunctional molecules, and natural products offer a potentially advantageous approach to therapeutics to remodel aggregation, their evaluation requires new platforms that are informed at the molecular level. To that end, we chose pulsed hydrogen/deuterium exchange mass spectrometry (HDX-MS) to discern the phenomena of aggregation modulation for a model system of alpha synuclein (αS) and resveratrol, an antiamyloid compound. We invoked, as a complement to HDX, advanced kinetic modeling described here to illuminate the details of aggregation and to determine the number of oligomeric populations by kinetically fitting the experimental data under conditions of limited proteolysis. The misfolding of αS is most evident within and nearby the nonamyloid-β component region, and resveratrol significantly remodels that aggregation. HDX distinguishes readily a less solvent-accessible, more structured oligomer that coexists with a solvent-accessible, more disordered oligomer during aggregation. A view of the misfolding emerges from time-dependent changes in the fractional species across the protein with or without resveratrol, while details were determined through kinetic modeling of the protected species. A detailed picture of the inhibitory action of resveratrol with time and regional specificity emerges, a picture that can be obtained for other inhibitors and amyloid proteins. Moreover, the model reveals that new states of aggregation are sampled, providing new insights on amyloid formation. The findings were corroborated by circular dichroism and transmission electron microscopy.

Keywords: amyloid formation; kinetic modeling of aggregation; limited proteolysis; modulator of alpha-synuclein; protein aggregation; pulsed hydrogen/deuterium exchange; resveratrol.

Figure 1.

Narrow range mass spectra of peptide 5–17 (2+; MKGLSKAKEGVVA) during HDX without (black) or with resveratrol (w Resv; red) at selected time points during aggregation. The plots display mass shifts as compared to the control in the absence of D₂O (at the top (blue)). The isotopic envelopes exhibit a bimodal distribution shaded with gray (species₁) or without shading (species₂). The protected, slow exchanging population indicative of aggregation is shaded by gray and denoted as species₁, whereas the fast exchanging, initial population that gradually begins to deplete in abundance is without shading and referred to as species₂. The distinct isotopic profiles are marked with dashed lines, and the fit (continuous line) is the sum of the two components.

Figure 2.

Narrow range mass spectra of peptide 77–89 (2+; VAQKTVEGAGSIA) during HDX without (black) or with resveratrol (w Resv; red) at selected aggregation times. The spectral mass shifts are manifest when compared to an isotope distribution in the absence of D₂O (top, blue). The isotopic envelopes exhibit a binomial distribution shaded with gray (species₁) or without shading (species₂). The protected, slow exchanging population that reports on aggregation is shaded by gray and denoted as species₁, whereas the fast exchanging, initial population that gradually begins to deplete in abundance is without shading and refers to species₂.The outlines of the two distinct isotopic clusters are marked by dotted lines and the fit (continuous line) is the sum of the two profiles.

Figure 3.

Average fractions of species₁ and species₂ with standard deviation for peptides, 55–69 (2+), 55–75 (2+) and 77–89 (2+) residing near in the NAC region in the absence (left) or in the presence of resveratrol (w Resv; right) across the complete time series of aggregation. The protected, slow exchanging population is shown with red open circles and denoted as species₁, whereas the fast exchanging, initial species that decreases in abundance is shown by purple open circles and referred to as species₂. Corresponding fits derived from modeling (described later) are shown with solid lines.

Figure 4.

Model mass and number concentration plots from fitting as described in SI Part 1 to all the seven peptide species fraction data vs aggregation time. These model mass concentration trends are combined as specified in the Signal Table SI1 to form the model HDX species fractions to match the experiment HDX species fractions for each peptide as shown in Figure S6. The mass concentration of each modeled population is color coded as follows: M₁: dark blue dotted line, M₂: green dotted line, M₃: black dotted line, M₄: light blue dotted line, M₅: pink dotted line, M₆: orange dotted line, m* brown dotted line, m**: dark green dotted line, m: red dotted line and M_off: grey dotted line and the number concentrations corresponding to each population are shown with a continuous line in matching color.

Figure 5.

Model mass and number concentration plots from fitting as described in SI Part 2 to seven peptide species fraction data vs aggregation time and to seven peptide total signals vs aggregation time. These model mass concentration trends are combined as specified in the Signal Table SI5 to form the model HDX species fractions after accounting for proteolysis yields to match the experiment HDX species fractions for each peptide as shown in Figures S11 and S12. Each population mass concentration modeled is dotted and color coded as given in the legends. The number concentrations corresponding to each population are shown with a continuous line in matching color. The R suffix denotes binding to resveratrol. The lower left quadrant is left blank because there are no populations bound with resveratrol.

Figure 6.

Far-UV CD spectra of αS without or with of resveratrol (w Resv) at selected times of the aggregation. All far-UV CD represent average of seven scans after buffer subtraction.

Figure 7.

Transmission electron microscopy images of aS without or with resveratrol at selected times of aggregation. The electron micrographs were obtained by staining with 4% uranyl acetate. The scale bars represent 200 nm.